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LACTIC ACID FERMENTATION OF PASTEURIZED AND POWDERED MILK AND
OPTIMIZING THE FACTORS AFFECTING THE FERMENTATION PROCESS
A PROJECT SUBMITTED TO BRAC UNIVERSITY IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE BACHELORS DEGREE IN MICROBIOLOGY
SUBMITTED BY
NOWSHIN SHYARA SHARAR
STUDENT ID: 10326008
DEPARTMENT OF MATHMETICS AND NATURAL SCIENCES (MNS)
BRAC UNIVERSITY
DECEMBER 2013
DECLARATION
I hereby solemnly declare that the research work embodying the results reported in this project
entitled “LACTIC ACID FERMENTATION OF PASTEURIZED AND POWDERED MILK
AND OPTIMIZING THE FACTORS AFFECTING THE FERMENTATION PROCESS”
submitted by the signatory has been carried out under the supervision of Professor Dr. Naiyyum
Choudhury, Coordinator of Biotechnology & Microbiology Program, MNS Department, BRAC
University. This project work has been implemented in the Microbiology Laboratory of MNS
Department. It is further declared that the project work presented here is original and has not
been submitted to any other institution for any degree or diploma.
Nowshin Shyara Sharar
(Candidate)
Certified By
Professor Dr. Naiyyum Choudhury
Supervisor
Coordinator of Biotechnology & Microbiology Program
MNS Department, BRAC University, Dhaka-1212
~ DEDICATED TO MY PARENTS ~
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
ACKNOWLEDGEMENT
First of all, I would like to express my gratitude to the Almighty Allah for giving me the courage and will to
finish my whole work properly in spite of facing so many problems. I would like to give a special thanks to
my respective teacher and supervisor, Professor Naiyyum Choudhury for encouragement, inspiration and
proper guidance. He has always been very kind and generous to me in every situation. He gave me the best
suggestion in the best possible way for the betterment of my project work. My gratefulness also goes to all
the teaching assistants and masters’ students; especially, Aditi apu, Asma apu and Anamika apu for
sacrificing their valuable time to help me in various circumstances where I have faced difficulties and
confusions. I thank Rabbi Sir for his honest opinions and suggestions. I also like to thank my friends and
fellow classmates; Iftekhar, Sreoshee & Chinmay for their useful advices as well as their endless support
during my work in this project through the whole semester. Finally, I would like to express my immense
appreciation towards my parents for their patience and hope during the course of time.
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
ABSTRACT
Yogurt is a basic dairy product that has been consumed for centuries as a part of the diet, even when its
beneficial effects were neither fully known nor scientifically proven. With time, yogurt has been
continuously modified to obtain a product with better appeal and nutritional effects. In this study, both
spontaneous and inoculated fermentation of pasteurized and powdered milk was performed in order to
study the quality of the yogurts. In addition to that, bacteria were isolated from both types of yogurts and
their microscopic characteristics had been observed under the microscope. Several factors are required to
produce a good quality yogurt; such as, temperature, pH, NaCl concentration, liquid and powdered milk
concentration etc. These fundamental factors were optimized in such a way that the yogurt was produced
in a short incubation period and the efficiency of the fermentation process was enhanced due to optimal
conditions. Moreover, the quality and texture of the yogurt remained unchanged up to 5 days.
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
TABLE OF CONTENTS
CHAPTERS TITLE PAGE NO.
DEDICATION OF THE PAPER i
ACKNOWLEDGEMENT ii
ABSTRACT iii
TABLE OF CONTENT iv
LIST OF TABLES viii
LIST OF FIGURES ix
LIST OF ABBREVIATION & SYMBOLS x
CHAPTER - 1 INTRODUCTION 1-4
1.1 IMPORTANCE OF THIS PROJECT 1
1.2 OBJECTIVES OF THE PRESENT STUDY 2
1.2.1 Specific Objectives 2
1.3 RESEARCH HYPOTHESIS 3
1.4 SCOPE & LIMITATION OF THIS STUDY 3
1.5 EXPECTED RESULTS 4
CHAPTER - 2 LITERATURE REVIEW 5-17
2.1 LACTIC ACID FERMENTATION OF MILK 5
2.2 CLASSIFICATION YOGHURT 6
A. Set Yogurt 6
B. Stirred Yogurt 6
C. Drinking Yoghurt 7
D. Frozen Yoghurt 7
E. Concentrated Yoghurt 7
F. Flavored Yoghurt 7
2.3 PRODUCTION OF DIFFERENT TYPES OF YOGHURT 8
2.4 HEALTH BENEFITS OF YOGHURT 9
2.5 COMPOSITION AND NUTRITIONAL FACTS OF MILK 9
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
2.6 MICROBIOLOGY OF YOGHURT STARTER CULTURES 11
2.7 CHARACTERISTICS & IDENTIFICATION OF LACTIC
ACID BACTERIA (LAB)
12
(A) Lactobacillus delbrueckii subsp. bulgaricus 12
(B) Streptococcus thermophilus 12
(C) Lactobacillus acidophilus 13
(D) Bifidobacterium bifidium 13
2.8 FACTORS AFFECTING FERMENTATION OF MILK 14
2.8.1 The Effect of pH on Yoghurt 14
A. Change in pH from 6.6 to 6.0 15
B. Change in pH from 6.0 to 5.0 15
C. At pH 6.0 15
D. At pH 5.0 15
2.8.2 The Effect of Rheological Property of Yogurt 16
2.8.3 The Effect of Physical Properties of Yogurt 16
2.9 CURRENT STATUS OF YOGURT PRODUCTION IN
BANGLADESH
17
2.10 CURRENT STATUS OF YOGURT PRODUCTION IN THE
WORLD
17
CHAPTER - 3 MATERIALS AND METHODS 18-23
3.1 MATERIALS 18
3.2 REAGENTS & CHEMICALS 18
3.3 INSTRUMENTS 19
3.4 SPONTANEOUS FERMENTATION METHOD 19
3.5 INOCULATED FERMENTATION METHOD 20
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
3.5.1 Reviving Bacterial Culture 20
3.5.1.1 Inoculated Fermentation Using Bacterial Stock Culture 20
3.6 SELECTION OF OPTIMUM CONDITION FOR THE
FERMENTATION PROCESS
21
3.6.1 Effect of temperature 21
3.6.2 Effect of pH 21
3.6.3 Effect of NaCl concentration 21
3.6.4 Effect of Powdered Milk concentration in Liquid Milk 22
3.6.5 Effect of Powdered Milk concentration in Water 23
CHAPTER - 4 RESULTS 24-46
4.1 SPONTANEOUS FERMENTATION 24
4.2 INOCULATED FERMENTATION 26
4.2.B Inoculated fermentation using MRS Agar plate & MRS
Broth Culture
34
4.3 Effect of temperature 35
4.4 Effect of pH 36
4.5 Effect of NaCl concentration 38
4.6 Effect of powdered milk concentration in liquid milk 43
4.7 Effect of powdered milk concentration in water 44
CHAPTER - 5 DISCUSSION 47-49
5.1 SPONTANEOUS FERMENTATION 47
5.2 INOCULATED FERMENTATION 47
5.3 Effect of temperature 48
5.4 Effect of pH 48
5.5 Effect of NaCl concentration 49
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
5.6 Effect of powdered milk concentration in liquid milk 49
5.7 Effect of powdered milk concentration in water 49
CHAPTER - 6 CONCLUSION 50
CHAPTER - 7 REFERENCES 51
CHAPTER - 8 APPENDIX 54-59
APPENDIX - A 54
APPENDIX - B 58
APPENDIX - C 59
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
LIST OF TABLES
TABLE NO. TITLE PAGE NO.
A Health benefits when milk is fermented 10
1.1 Characteristics of yogurts made by spontaneous fermentation 24
1.2 Tabular presentation of microscopic characteristics of the cells 25
2.1 Characteristics of yogurts made by inoculated fermentation 26
2.2 Characteristics of yogurts made by inoculated fermentation 27
2.3 Tabular presentation of microscopic characteristics of the cells 29
2.4 Characteristics of yogurts made by inoculated fermentation 30
2.5 Tabular presentation of microscopic characteristics of the cells 31
2.6 Characteristics of yogurts made by inoculated fermentation 32
2.7 Tabular presentation of microscopic characteristics of the cells 33
3 Characteristics of yogurts made by using bacterial stock culture 34
4 Effect of temperature on lactic acid fermentation of milk 36
5 Effect of pH on lactic acid fermentation of milk 37
6.1 Effect of NaCl concentration on lactic acid fermentation of milk 39
6.2 Effect of NaCl concentration on lactic acid fermentation of milk 41
6.3 pH of milk measured at 1-hour intervals 41
6.4 Tabular presentation of microscopic characteristics of the cells 42
7 Effect of powdered milk concentration in liquid milk 44
8 Effect of powdered milk concentration in water 46
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
LIST OF FIGURE
FIGURE NO. TITLE PAGE NO.
1 Yogurts made by spontaneous fermentation 24
2 The cell morphology observed under a compound microscope 25
3 Yogurts inoculated from Shakti & Ultra Doi
26
4 Sub-cultured from spontaneously fermented yogurt (fig.1) &
Ultra doi (fig.2)
27
5 The cell morphology observed under a compound microscope 28
6 Continuous fermentation process
30
7 The cell morphology observed under a compound microscope 31
8 Yogurts after 5 and 6 hours of incubation period at 37˚C 32
9 The cell morphology observed under a compound microscope 33
10 Inoculated fermentation of milk using bacterial culture from
MRS agar plate
34
11 Effect of temperature at 45˚C (a), 55˚C (b) & 60˚C (c & d)
respectively
35
12 Effect of pH 5.5 (a), pH 6.5 (b) and pH 8.5 (c) respectively 37
13 Effect of NaCl concentrations (0.1 %, 0.5 % and 1.0 %) 38
14 Yogurts inoculated from 0.1 % NaCl conc. yogurt previously
prepared
40
15 The cell morphology observed under a compound microscope 42
16 Effect of powdered milk conc. (2%, 4%, 6%, 8% and 10%) in
liquid milk
43
17 Effect of powdered milk conc. (2%, 4%, 6%, 8% and 10%) in
water
45
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
LIST OF ABBREVIATION & SYMBOL
SERIAL NO. TITLE ABBREVIATION
1 HCl Hydrochloric Acid
2 NaCl Sodium Chloride
3 NaOH Sodium Hydroxide
4 LAB Lactic Acid Bacteria
5 MRS de Man Rogosa Sharpe
6 Ca, Na Calcium, Sodium
7 sp. Species
8 ˚C Degree Celsius
9 Kg, g, ml Kilogram, gram, Milliliter
10 et al And friends
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
INTRODUCTION
1.1 IMPORTANCE OF THIS STUDY
Yogurt (also spelled yoghurt) is a semisolid fermented milk product that originated centuries ago and
has evolved from many traditional Eastern European products. It is eaten all over the world. The partial
digestion of the milk caused by the fermentation of the starter culture makes yogurt easily digested, even
by people who cannot tolerate milk. It is a rich source of protein and calcium, and the fermentation
process makes these nutrients easier to absorb.
All yogurts must contain at least 8.25% solids not fat. It can be made from ready-made yogurt or starter
culture. The main (starter) cultures in yogurt are Lactobacillus bulgaricus and Streptococcus
thermophilus. The function of the starter cultures is to ferment lactose (milk sugar) to produce lactic
acid. The increase in lactic acid decreases pH and causes the milk to clot, or form the soft gel that is
characteristic of yogurt. The fermentation of lactose also produces the flavor compounds that are
characteristic of yogurt. The acidic condition also restricts the growth of food poisoning pathogens and
some spoilage bacteria. So, whereas milk is potential source of food poisoning and only has a shelf life
of few days, yogurt is safer and can be kept for up to ten days, under proper storage conditions.
The secret to tasty yogurt is in the proper control of the temperature at various stages. If the temperature
is too low, the culture grows too slowly to adequately acidify milk and to achieve a good texture. In
addition, there is a subtle difference in the taste because the formation and secretion of metabolites
which contribute to the overall taste are dependent on the growth rate. The window of proper
fermentation is quite small, i.e. from 37 ºC to 44 ºC. In general, as the temperature is raised up to 44 ºC,
the rate of culture metabolism is higher, and the yogurt is sweeter. Faster growth also prompts the yogurt
to set faster. When the desired acidity is reached, yogurt is quickly cooled to halt further fermentation
and metabolic activity. This cooling step must be done quickly to control the acidity of the yogurt,
which has a profound effect on the taste.
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
Except for L. bulgaricus and S. thermophilus, other bacterial cultures, such as Lactobacillus
acidophilus, Lactobacillus subsp. casei, and Bifido-bacteria may be added to yogurt as probiotic
cultures. Probiotic cultures benefit human health by improving lactose digestion, gastrointestinal
function, and stimulating the immune system. Ongoing studies are revealing further health benefits to
this great food, such as reducing yeast infections and colon cancer.
1.2 OBJECTIVE OF THE PRESENT STUDY
The main objective of this project work is to observe the two different methods of fermentation process;
e.g. spontaneous and inoculated fermentation method and studying the necessary factors affecting the
fermentation process. There are many aspects which are very crucial for carrying out a fermentation
process. Among them temperature, pH, NaCl concentration, liquid and powdered milk concentration are
the most basic and fundamental factors. These conditions will be optimized to improve the fermentation
of milk and produce a fine quality yogurt with a smooth texture and firm consistency. Therefore, the
factors affecting the overall method will be thoroughly studied and optimized in the laboratory to
accomplish a enhanced fermentation process.
1.2.1 Specific Objectives
The specific objectives of this study are as follows:
Spontaneous fermentation method
Inoculated fermentation method (from commercial yogurt & stock culture of LAB)
The cell morphology of bacteria
The effect of temperature
The effect of pH
The effect of NaCl concentration
The effect of powdered milk concentration in liquid milk
The effect of powdered milk concentration in water, etc.
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
1.3 RESEARCH HYPOTHESIS
In milk fermentation, lactic acid bacteria produce lactic acid, reduce the pH and therefore coagulate the
milk. Desirable strain of lactic acid bacteria (LAB) can be added to the fermentation process. Similarly,
live cultures from commercial yogurt sample can also be used as a starter culture for the fermentation
process. In order to yield a good quality yogurt, the various factors necessary to carry out fermentation
can be adjusted to improve the overall process. If the physiochemical properties; e.g. temperature, pH
and concentration of both liquid and powdered milk are increased or decreased, the quality and flavor of
the yogurt will be enhanced. Moreover, if the incubation period is reduced into 5-6 hours from 18-24
hours; the bacteria will grow more rapidly and ferment the milk sample in a shorter period of time to
produce yogurt.
1.4 SCOPE & LIMITATION OF THIS STUDY
In spontaneous fermentation, bacteriophage may contaminate the milk and alter the quality of yogurt.
On the other hand, in inoculated fermentation, undesirable smell and off-flavor may come from the
yogurt if the starter cultures are of poor quality or become contaminated. For this reason, fresh, recently
purchased culture or freshly grown bacterial culture should be used each time to make yogurt. Adding
culture to very hot milk (>115°F) can kill bacteria; this is why thermometer should be used to carefully
control the temperature of milk. Too hot or too cold of incubation temperature can slow down culture
growth; so the temperature of the incubator must be adjusted beforehand.
Yogurt generally has a 10-21 day shelf life when made and stored properly in the refrigerator below
40°F (4.4˚C). Molds, yeasts and slow-growing bacteria can spoil the yogurt during prolonged storage.
Ingredients added to yogurt should be clean and of good quality. Introducing microorganisms from
yogurt add-ins can reduce shelf life and result in quicker spoilage. Yogurt samples with visible signs
of microbial growth or any odors other than the acidity of fresh yogurt should be discarded
straightaway. Whey collects on the surface of the yogurt. This process is called syneresis. Some
syneresis is natural. Excessive separation of whey, however, can be caused by incubating yogurt for
too long or by agitating the yogurt while it is setting. Moreover, if the yogurt has over-set or incubated
too long; it must be refrigerated immediately after a firm coagulum has formed.
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
1.5 EXPECTED RESULTS
In this study, both spontaneous and inoculated fermentation method of milk fermentation will be
performed in the laboratory. Live cultures of lactic acid bacteria (LAB) will be taken and used from
both commercial yogurt and stock culture. The major physicochemical properties (temperature, pH,
NaCl concentration, powdered milk concentration etc.) affecting the fermentation process will be
optimized to enhance the fermentation performance. Additionally, a shorter incubation period for the
lactic acid fermentation of pasteurized and powdered milk samples will be determined in order to
produce yogurt in a small period of time. The findings of this study are expected to contribute to the
production of a good quality yogurt.
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
CHAPTER: 02
LITERATURE REVIEW
2.1 LACTIC ACID FERMENTATION OF MILK
Yoghurt is a fermented milk product obtained from milk by the lactic acid fermentation through the
action of Streptococcus salivarius subsp. thermophiles and Lactobacillus delbrueckii subsp. bulgaricus
(FAO/WHO, 1977). When a sufficient quantity of lactic acid is produced, the milk coagulates and this
coagulated milk is called yogurt. The word “yoghurt” is derived from Turkish “jugurt”, used to describe
any fermented food with an acidic taste (Younus et al., 2002). Historically, yoghurt was made by
fermenting milk with indigenous microorganisms. Yoghurt having high nutritional and therapeutic
properties is being highly consumed and produced (Karagul et al., 2004). Yoghurt is stored at 2- 4ºC
throughout the distribution chain for avoiding risk of spoilage from yeasts (Tamime et al., 2000) and
also for preventing further activity by starter culture. The probiotic yogurt, having probiotic effect is a
fermented milk product with adjuvant microorganisms. A high population of probiotic organisms in the
colon contributes to good intestinal health. Consequently consumption of products such as yogurt
containing viable probiotic organisms adds benefit to human gut health. Moreover, yogurt is an excellent
source of calcium, phosphorus, potassium and contains significant quantities of general vitamins.
Yoghurt could be used for feeding, owing to its higher Ca/Na ratio (Demott, 1985). Yogurts vary in
appearance, flavor and ingredients. The quality and composition of yoghurt of applied bacterial cultures
affects the quality of the yoghurt obtained as a result of the milk fermentation processes. There is a
symbiotic relationship between the two species of bacteria, Lactobacillus bulgaricus and Streptococcus
thermophiles and so there is more rapid acid development than in single strain culture (Rasic et al.,
1978; Tamime et al., 1980). Various combinations of starter cultures are selected during manufacturing
of yoghurt to achieve desirable characteristics of product and to provide the consumers with a wide
choice of therapeutic benefits. Depending on its activity, manufacturer usually adds 2-4 % yogurt starter
culture. Now a days, there has been an increasing trend to fortify the dairy product with fruits (natural
fruit juice, pulp, dry fruits) (Desai et al., 1994; Ghadge et al., 2008). Aesthetic value of new product can
be increased by using fruit juice as a functional pigment in the fermented milk
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
with an array of colors and flavor properties (Coisson et al., 2005). Euterpeoleracea juice can be used as
functional pigment for yoghurt, which is dark purple in color having high anthocynin and phenolic
content (Coisson et al., 2005). Yogurt is a functional food. The functional food includes probiotics,
prebiotics and synbiotics. Probiotics can be defined as “live microbial feed supplements that beneficially
affect the host animal by improving its intestinal microbial balance” (Champagne et al., 2005). The
“non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth
and/or activity of one or a limited number of bacteria in the colon”. Symbiotic is a combination of
probiotics and prebiotics that “beneficially affects the host by improving the survival and the
implantation of live microbial dietary supplements in the gastro-intestinal tract by selectively stimulating
the growth and/or by activating the metabolism of one or a limited number of health promoting bacteria”
(DiRienzo et al., 2000).
Yogurt provides two significant barriers against pathogen growth; e.g. heat and acidity (low pH). Both
are necessary to ensure a safe product. Acidity alone has been questioned by recent outbreaks of food
poisoning by E. coli O157:H7 that is acid-tolerant. E. coli O157:H7 is easily destroyed by pasteurization
(heating). Therefore, milk sample must be boiled before making yogurt at home or commercially
pasteurized milk should be used to make yogurt.
2.2 CLASSIFICATION YOGHURT
There are different types of yoghurt produced worldwide. However particular yogurt may be subdivided
into different groups based on the following aspects:-
A. Set Yogurt
This type of yoghurt is incubated and cooled in the final package and is characterized by a firm jelly like
texture. Set yoghurt is fermented in a retail container, filled after milk inoculation and is incubated in an
incubation room at a suitable temperature normally 40- 43°C for approximately 2 hours and 30 minutes
to 4 hours (Desai et al., 1994).
B. Stirred Yogurt
This type of yoghurt is incubated in a tank and the final coagulum is "broken" by stirring before cooling
and packing. The texture of stirred yoghurt will be less firm than a set yoghurt somewhat like a very
thick cream. A little reformation of coagulum will occur after packaging. Stirred yogurt is a non-
Newtonian fluid, obtained by promoting the growth of Lactobacillus delbrueckii
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus at a mild temperature (between
40°C and 43°C) until a desired acidity level is reached. In stirred yoghurt, milk is inoculated and
incubated in a fermentation tank, the yoghurt gel being broken up during the stirring, cooling and packaging
stages. Due to several factors there may be variations in the rheological properties of stirred yoghurt. These can be
of a physical nature such as those related with total solid content, milk composition and type of starter culture or
processing conditions; such as, homogenization, thermal pre-treatment of the milk and post-incubation stages
(including: stirring, pumping, cooling and packaging) (Tamime et al., 1980).
C. Drinking Yoghurt
It also has the coagulum "broken" prior to cooling. In drinking yoghurt, the agitation used to break the
coagulum is severe. Very little reformation of coagulum may occur.
D. Frozen Yoghurt
Frozen yoghurt is inoculated and incubated in the same manner as stirred yoghurt. However, cooling is
achieved by pumping through a chiller or freezer in a fashion similar to ice-cream. The texture of the
finished product is mainly influenced by the freezer and the size and distribution of the ice crystals are
produced.
E. Concentrated Yoghurt
This type of yoghurt is inoculated and fermented in the same manner as stirred yoghurt. Following the
breaking of the coagulum, the yoghurt is concentrated by boiling off some of the water, which is often
done under vacuum to reduce the temperature required. Heating of low pH yoghurt can often lead to
protein being totally denatured and producing rough and gritty textures. This is often called strained
yoghurt due to the fact that the liquid that is released from the coagulum upon heating used to strain-off
in a manner similar to making soft cheese.
F. Flavored Yoghurt
The flavors are usually added at or just prior to filling into pots. Common additives are fruit or berries,
usually as a puree or as whole fruit in syrup. These additives often have as much as 50% sugar in them;
however, with the trend towards healthy eating gaining momentum; many manufacturers offer a low
sugar and low fat version of their products. Low or no sugar yoghurts are often sweetened with
saccharin or more commonly with aspartame.
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
2.3 PRODUCTION OF DIFFERENT TYPES OF YOGHURT
Flowchart: Showing Processing of Yoghurt
Flowchart: Showing Processing of Set and Stirred Yoghurt (W.J Lee and J.A Lucey, 2010)
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
2.4 HEALTH BENEFITS OF YOGHURT
Probiotic yoghurt is aimed at reducing medical conditions; such as constipation and diarrhea by
restoring the beneficial microbial population in the colon.
It is beneficial to our digestive system, especially stomach and colon. Cow’s milk is preferred for
preparing yoghurt as having low fat.
It provides immunity; protect us from cold, cough and strengthen body’s defense mechanism.
It strengthens the collagen in the skin and is good for our skin.
It lowers the blood pressure, bad cholesterol and risk of heart attacks.
Yoghurt is a source of natural proteins; it is safer for those having problem in tolerance of
lactose. Yoghurt is rich in calcium; so it protects the bones against osteoporosis and arthritis.
It discourages vaginal yeast infections.
It helps in cutting down calorie and thus helps in burning fat.
By daily consumption of yoghurt, disease causing bacteria are flushed out from the colon and
therefore, help in protecting against colon cancer.
Consumption of yoghurt can shut down Helicobater pylori; the bacterium responsible for most
peptic ulcers.
2.5 COMPOSITION AND NUTRITIONAL FACTS OF MILK
Nutrients in milk can differ depending on the age, breed of cattle or goat, the season of the year and the
food it is fed or grass the animal grazes. On average, milk has 4% fat, 3.4% protein and 4.8% milk
sugar (lactose) and provides approximately 730 kcal in energy per liter. As such, a serving of 250 ml
glass would therefore provide 180 kcal, which is 10% of an under five year old child’s energy needed
for a day and 6% of a symptomatic HIV positive adult’s energy needed. Milk also provides valuable
vitamins; e.g. vitamin A (for eyes, skin, hair, mucous membranes and immunity), vitamin B’s (important
for metabolism of carbohydrates in foods to produce energy) and vitamin D (important for strong bones
and teeth), as well as minerals, calcium and phosphorus; both of which are important for strong bones.
(Source: http://www.fao.org/docrep/v1650t/v1650T0s)
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
Table A: Showing Health benefits when milk is fermented
Effect of fermentation Changes in milk Effects on health
Increase in lactic acid bacteria
(LAB) population
Reduced lactose content in
milk
Reduced number of
pathogenic bacteria
No diarrhea and bloating
Improved gut health
Prevention and protection
from bacterial vaginitis
and fungal infections in
women
Ability to digest remaining
lactose in the fermented
milk and use as an energy
source
Breakdown to short chain
peptides
Identification of casein
peptides and whey peptides
with functional properties
Easier digestion
Some with
antihypertensive effects
Some with pain relief
effects
Some with immune
enhancing properties
Some with calcium
binding bone building
properties
Increased acidity Sharpness in taste Acidity prevents pathogenic
bacterial growth in milk
*Reference for changes in energy requirements in HIV: ‐ http://whqlibdoc.who.int/publications/2003/9241591196.pdf
*Reference for energy requirements in the lifecycle: ftp://ftp.fao.org/docrep/fao/007/y5686e/y5686e00.pdf
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
2.6 MICROBIOLOGY OF YOGHURT STARTER CULTURES
There is a symbiotic relationship between the two species of bacteria i.e. Lactobacillus bulgaricus and
Streptococcus thermophiles that’s why there is more rapid acid development than in the single strain
culture (Rasic et al., 1978; Tamime et al., 1980).
Figure: Showing symbiosis between Streptococcus thermophilus and Lactobacillus delbrueckii Subsp.
bulgaricus.
As shown in the above figure, Streptococcus thermophilus produces lactic acid and small quantities of
formic acid, which promotes outgrowth of Lactobacillus delbrueckii Subsp. bulgaricus. On the other
hand, Lactobacillus delbrueckii subsp. bulgaricus species produce amino acids to stimulate the growth
of Streptococcus thermophilus Chemical composition of the milk base (total solids and fat content) have
significant effects on the activity of starter cultures. Ozer and his co-worker have studied the behavior of
starter cultures in concentrated yoghurt produced by different methods. They discovered that production
method and total solid content in milk that influenced the growth and activity of starter cultures (Ozer
and Robinson, 1999).
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
2.7 CHARACTERISTICS & IDENTIFICATION OF LACTIC ACID BACTERIA (LAB)
(A) Lactobacillus delbrueckii subsp. bulgaricus
It is a gram-positive rod shaped filamentous, non-motile, non-spore forming bacteria. It requires a low
pH (5.4 to 4.6) to grow effectively so the bacterium is regarded as acidouric or acidophilic. The
bacterium has complex nutritional requirements including the inability to ferment any sugar except
lactose, from which it produces lactic acid that helps to preserve yoghurt. It is often helpful to sufferers
of lactose intolerance whose digestive systems lack the enzymes to break down lactose to simpler
sugars. While fermenting milk, it produces acetaldehyde, which is one of the main yoghurt aroma
components.
Figure: Microscopic observation of bacterial cells (source: www.bacterianamehere.pbworks.com)
(B) Streptococcus thermophilus
The genus Streptococcus comprises several harmful pathogenic species such as Streptococcus
pyogenesor, Streptococcus pneumoniae, together with a single, “Generally Recognized As Safe
species”- S. thermophiles and S. thermophilus are widely used for the manufacture of dairy products
(Cvetan, 1998; Fox, 1993).
Figure: Microscopic observation of bacterial cells
(Image source: http://www2.unibas.it/parente/Starter/gruppi.html)
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
(C) Lactobacillus acidophilus
Lactobacillus acidophilus shows a range of health benefits which include: providing immune support for
infections and cancer, reducing occurrence of diarrhea in human, aiding in lowering cholesterol,
improving the symptoms of lactose intolerance. Studies have shown that dietary supplementation with L.
acidophilus decrease the number of colon cancer cells in a dose dependent manner (Rao, 1999). The
better survival of L. acidophilus when added along with the starter was presumably due to the transient
tolerance of the culture to hydrogen peroxide (Speck, 1976).
Figure: Microscopic observation of bacterial cells
(D) Bifidobacterium bifidium
Bifidobacteria are one of the most important group of intestinal organisms with regard to human
health.Bifidobacterium spp. belongs to the dominant anaerobic flora of the colon. The main species
present in human colon are Bifidobacteriumadolescentis, B. bifidum, B. longumsubsp.infantis, B.
breveand B. longum (Holzapfel, 2001). B. bifidum was found to be tolerant to the acidity of a model
gastrointestinal tract system, with only a 20 % decrease in numbers as the pH decreased from 5.0 to 1.8
over an 80 min period (Holzapfel, 2001). Important property of probiotic bifidobacteria is acid
tolerance, enabling the cells to survive gastric acidity and volatile fatty acids produced during
fermentation in the intestine (Charteris, 1998). In the presence of yoghurt starter organisms the growth
of bifidobacteria seems to be suppressed. Bifidobacteria have a higher resistance to acid and bile present
in the gastrointestinal tract than yoghurt bacteria. The ingestion of bifidus fermented milk led to an
increase in total bifidobacteria which was related to the colonic transit of the exogenous bifidobacteria
(Bouhnik, 1996). In yoghurt like product, addition of probiotic cultures to the normal starters generally
results in slower growth of the probiotic strains than if they were added alone in milk (Roy, 1996;
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
Samona, 1994). These starters produce environments that inhibit the growth of not only pathogens and
spoilage microorganisms but also of probiotic (Vinderola, 2002). The phenomenon could partially be
related to the production of bacteriocins or other inhibitors such as lactic and other organic acids and
hydrogen peroxide produced by the starter cultures (Maus, 2003; Vinderola, 2000). In addition, starter
cultures grow faster, acidification occurs rapidly, and fermentation times are much shorter in their
presence which resulted in reduced availability of nutrients (Shah, 2000); thus probiotic cultures do not
have time to grow extensively. Many authors also reported the capacity of bifidobacteria to synthesize
galacto-oligosaccharides (Hung and Lee, 2002; Lamoureux, 2002; Saxelin, 1999).
Figure: Microscopic observation of bacterial cells
(Image source: http://barefootprovisions.com)
2.8 FACTORS AFFECTING FERMENTATION OF MILK
There is several factors necessary milk fermentation process. Some of them are described below:
2.8.1 The Effect of pH on Yogurt
The ideal pH of the finished product of yogurt should be 4 to 4.1. However this pH also depends on
adding fruits or flavoring agent to the yogurt. After fermentation when the yogurt is ready to eat, the pH
should be 4. The pH of yoghurt is decreased if the amount of skimmed milk powder is increased. The
yogurt mixture coagulates during fermentation due to the drop in pH. The streptococci are responsible
for the initial pH drop of the yogurt into approximately 5.0. The Lactobacillus species are responsible
for further decrease in pH into 4.5.
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
Shaker et al studied the effect of milk fat content on the acid development during fermentation and
rheological properties of plain yoghurt. They indicated that increasing milk fat content increased the
initial pH of the samples and the rate of decreasing pH during incubation of high fat samples was lower
than others (Shaker et al, 2000).
A. Change in pH from 6.6 to 6.0
When the pH of the milk is decreased from 6.6 to 6.0 then the electrostatic repulsion decreases; as the
net negative charge on the casein micelles decreases. The size of the casein micelles remains unchanged
as the small amount of colloidal calcium phosphate is solubilized at pH > 6.0
B. Change in pH from 6.0 to 5.0
When the pH changes from 6.0 to 5.0 then there is decrease in electrostatic repulsion and steric
stabilization occurs which are responsible for the stability of casein micelles in the original milk. Due to
the decrease in the pH, the negative charge on casein micelles decreases and the charged tails of casein
curls up or may shrink.
C. At pH 6.0
At this pH, the electrostatic repulsion between the exposed phosphoseriene residues increases due to the
increase in the rate of solubilization of colloidal calcium phosphate, which weakens the internal
structure of casein micelles.
D. At pH 5.0
The decrease in electrostatic repulsion between casein molecules occurs at pH 5; as the net negative
charge on the casein decreases. When the pH of milk becomes close to the isoelectric point (pH 4.6)
then casein-casein attractions is amplified due to increased hydrophobic and electrostatic charge
interactions (Horne, 1998). The three dimensional network is formed consisting of clusters and chains of
caseins due to the acidification process (Mulvihill and Grufferty, 1995).
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
2.8.2 The Effect of Rheological Property of Yogurt
Food rheology is the study of the deformation and flow of food materials (Rao, 1999). It can be
classified as a pseudoplastic material (contains a yield stress that has to be exceeded for flow to be
initiated) that can be either a viscoelastic fluid (stirred yoghurt) or viscoelastic solid (set yoghurt).
Viscoelastic indicates that the material is having some of the elastic properties of an ideal solid and
some of the flow properties of an ideal (viscous) liquid. Yogurt exhibits time-dependent shear thinning
behavior but is not a true thixotropic material, since structural breakdown due to shear is not completely
reversible once the shear stops.
2.8.3 The Effect of Physical Properties of Yogurt
It has been found that addition of protein (i) improves water holding capacity (ii) decreases the syneresis
effect and (iii) stabilizes the pH of the samples during storage (Athanasios G. Stefanakis et al). The
physical and sensory properties of yoghurt gels are highly influenced by the protein content and the total
solids content of the yoghurt milk. A study on the effect of milk supplementation (whey, casein
hydrolysate and milk protein) on the acidification and microbiological stability of fermented milks
showed that acidifying activity was greatly improved with casein hydrolysate, with a reduction of the
fermentation time by 55% in comparison with the other supplementation (Oliveria et al., 2001).
Abd-El Salam et al., (1982) showed that total solids content had no adverse effect on starter activity or
coagulation time. Increasing milk total solids from 16 to 23% had significant effect on decreasing rate of
pH during fermentation (Ozer et al., 1998). The incubation time for the milk at pH 4.6 was shorter than
the time of retentive (Tamime et al., 1989). The increase in milk fat content influences the growth and
activity of starter cultures in samples with two levels of total solid (12 and 23%) (Mahdian et al., 2007).
The chemical composition of the milk base especially total solids has the major effect on the
acceptability of concentrated yoghurt. Concentrated yoghurt containing less than 20% total solid was
assessed as thin and tasteless whereas, greater than 25% total solid became gummy and bitter (Robinson,
1977).
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
2.9 CURRENT STATUS OF YOGURT PRODUCTION IN BANGLADESH
In Bangladesh, curd (Dadhi) is one of the most popular fermented milk products which are made in mud
pots, usually from cow’s milk, sometimes from buffalo milk. The bacteria used are Lactobacillus
bulgaricus, Lactobacillus plantarum and Streptococcus thermophiles and/or Lactotococcus lactis. In
making curd, the inoculum is higher than is usual for other fermented milk products (approximately 5%-
8%). The traditional fermented dahi is prepared without any care of quality control and hygienic
conditions and contain a lot of contaminants which may not be friendly to the body; it still enjoys loyal
following in rural communities in Bangladesh and a must dessert after an improved heavy meal. In
addition to poor quality control and hygiene during manufacturing, dahi are sold almost in every open
market and products are kept on shelf at ambient temperature without any covering while very few
retailers keep their product in refrigeration. Therefore, periodical inspection on the quality of the product
in necessary, then again is not in practice. Very few researchers are conducting the study of market dahi
quality on region basis and considering the investigation upon renowned brand dahi of Bogra, Muslim,
Bonoful, Aarong and Grameen Soktee* to check and compare their quality with the dahi prepared from
fresh raw milk by following standard procedures (Bhattahacharya, K.P., 1980).
2.10 CURRENT STATUS OF YOGURT PRODUCTION IN THE WORLD
Alternative methods to improve quality of low-fat yoghurt become an area of considerable research
interest. Yoghurts that have past their ‘best before’ date constitute a waste that has to be environmentally
treated. It can be used as a source for lactic acid production by Lactobacillus casei. Production of
yoghurt generates a residue highly supplemented with sugar and fruits syrups that can be metabolized to
lactic acid. Rheological properties were not seriously affected by freezing, while the drying step resulted
in an overall structural weakening of the reconstituted products, possibly as a consequence of the
mechanical energy required for mixing with water. However, its viscoelastic properties were retained
and the original strength could be recovered by modulating the amount of water. Mortality levels of
bacterial population were reduced when sucrose and blueberries were added as ingredients. Addition of
herbs or their active components like oils could be an effective strategy to improve functionality of milk
and milk products with respect to the health benefits, food safety and bio preservation. These
components should improve therapeutic quality without any adverse effect on sensory and rheological
attributes.
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
CHAPTER: 03
MATERIALS & METHODS
3.1 MATERIALS
Aarong Full-cream Pasteurized Milk (1 liter)
Milk Vita Pasteurized Milk (1 liter)
Dano Milk Powder (1000 g)
Ultra Doi
Shakti Doi
Bacteria Stock Culture
Lactobacillus MRS agar
Lactobacillus MRS Broth
Distilled water
3.2 REAGENTS & CHEMICALS
Name of the reagents
0.85% Normal saline Crystal Violet
1 M NaOH Gram’s Iodine
32% HCl Safranin
Ethanol NaCl
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
3.3 INSTRUMENTS
Name of the instruments
Laminer Airflow Vortex mixture
Compound Microscope Refrigerator
Autoclave Balance machine
Incubator Spirit lamp
3.4 SPONTANEOUS FERMENTATION METHOD
200 ml Milk Vita pasteurized milk was taken in a 250 ml beaker and boiled. Then the milk was cooled
to room temperature and 100 ml milk was added to two separate plastic containers. No inoculum or
bacterial culture was added into the containers. After that, the containers were incubated at 36˚C for 20
hours.
Smears were prepared on glass slides taking loopful of yogurt from each of the spontaneously fermented
milk containers and Grams’ staining was performed. The microscopic characteristics of the presumptive
organisms were observed under a compound microscope at 100X magnification.
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
3.5 INOCULATED FERMENTATION METHOD
In inoculated fermentation method, live culture of bacteria was used to ferment milk.
100 ml “Milk Vita” milk was boiled and cooled to room temperature. 1 teaspoon of yogurt from
the spontaneously fermented milk container was added into milk poured in the plastic containers.
They were incubated at 37˚C for 18 hours.
200 ml milk was boiled and cooled to room temperature. 1 teaspoon of yogurt from “Shakti Doi”
and 1 teaspoon of yogurt from “Ultra Doi” was taken and added into 100 ml milk poured into
each of the plastic containers. They were incubated at 45˚C for 22 hours.
Inoculum was taken separately from these set of experiments and inoculated into fresh “Milk Vita”
pasteurized milk. Likewise, this process was done three times by taking 1 tsp. of yogurt from each batch
and inoculated into fresh “Milk Vita” pasteurized milk separately, until pure culture was obtained from
the yogurts. In addition, the incubation period was shortened in each fermentation batch to an optimum
period of time (from 22 hours to 5 hours). Besides the fermentation process, the microscopic
characteristics of the presumptive organisms present in the fermented yogurts were also observed in
parallels.
3.5.1 Reviving Bacterial Culture
Five different bacterial strains were taken which were named S1, S2, LP, Aarong, LAB and streak plated
on to MRS agar medium and incubated at 37˚C for 48 hours. Isolated colonies were transferred to MRS
broth and incubated at 37˚C for 48 hours. Since the bacterial growth was slow, the plates and tubes were
kept more than 24 hours in the incubator.
3.5.1.1 Inoculated fermentation using Bacterial Stock Culture
A loopful of bacterial colony from MRS agar plate and 10 ml culture from MRS broth tube were added
to 100 ml “Milk Vita” pasteurized milk (after boiling and cooling) in each container and stirred well.
The containers were incubated at 37˚C for 24 hours. After 24 hours, the physical appearances of the
yogurts were observed and recorded in a table.
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
3.6 SELECTION OF OPTIMUM CONDITION FOR THE FERMENTATION PROCESS
For selecting the optimum condition for lactic acid fermentation of milk several factors are associated.
The effect of temperature, pH, NaCl concentration, powdered milk concentration in water and liquid
milk were conducted to determine an optimum condition for the fermentation process in order to
produce a good quality yogurt.
3.6.1 Effect of temperature
To optimize the fermentation temperature, fermentation was carried out at 45˚C, 55˚C and 60˚C. All
other conditions were kept constant. The initial pH of the milk was measured 6.5 after calibrating the pH
meter with pH 7.0. 100 ml Aarong full-cream pasteurized milk was taken in each container and a
thermometer was used to carefully control the temperature of milk before adding the inoculum.
3.6.2 Effect of pH
To study the effect of pH on lactic acid fermentation of pasteurized milk, the fermentation process was
carried out at pH 4.5, 5.5, 6.5 and 8.5 in a volume of 100 ml Aarong full-cream milk in each container to
find out the optimum pH. While there were variations in pH, all other conditions were held constant.
The initial pH of the milk was measured 6.5 after calibrating the pH meter with pH 7.0. 32% HCl was
added drop by drop to reduce the pH into 4.5 and 5.5 respectively and 1 M NaOH was used to increase
the pH to 8.5. 1 tsp. of inoculum was added to each milk containers. The containers were incubated at
37˚C for 5 hours. The quality and texture of the yogurts as well as their final pH were recorded in a
table.
3.6.3 Effect of NaCl concentration
To study the effect of NaCl concentration for yogurt production, the fermentation process was carried
out with an initial pH 6.5 of Aarong full-cream pasteurized milk. At first, the 300 ml milk was measured
by a measuring cylinder and then boiled. After cooling, 100 ml milk was taken into three separate plastic
containers. To find out the optimum concentration of NaCl, three different concentrations of NaCl (0.1
%, 0.5 % and 1.0%) were prepared and added to the milk containers. 1 tsp. of inoculum was added to
each container and they were incubated at 37˚C for 5 hours. While there were variations in NaCl
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
concentrations, all other conditions (milk volume, inoculum size, pH & temperature) were kept constant.
After 5 hours of incubation, the quality and texture of the yogurts at different NaCl concentrations were
observed and recorded in a table.
After determining the optimum concentration of NaCl in yogurt production, 1 tsp. was taken from the
corresponding yogurt container and transferred to five containers having 100 ml freshly boiled Aarong
full-cream pasteurized milk. All of them contained 0.1 % NaCl concentration and they were incubated at
37˚C for 5 hours. The changes in pH of one of the five containers were measured at 1-hour intervals up
to 5 hours. The quality and texture of the yogurts at different NaCl concentrations were observed and
recorded in a table.
Furthermore, smears were prepared on glass slides taking loopful of the watery portion of yogurt from
each of the five containers and Grams’ staining was performed. The glass slides were placed under a
compound microscope and the microscopic characteristics of the presumptive organisms were observed
under 100X magnification.
3.6.4 Effect of Powdered Milk concentration in Liquid Milk
To study the effect of powdered milk concentration in yogurt production, the fermentation process was
carried out with an initial pH 6.5 of Aarong full-cream pasteurized milk. At first, the 500 ml milk was
measured by a measuring cylinder and then boiled. After cooling, 100 ml milk was taken into five
separate plastic containers. To find out the optimum concentration of powdered milk, five different
concentrations of Dano powdered milk (2%, 4%, 6%, 8% and 10%) were prepared and added to the
containers having liquid milk and dissolved properly. 1 tsp. of inoculum was added to each container
and they were incubated at 37˚C for 5 hours and 30 minutes. While there were variations among
powdered milk concentrations, all other conditions (milk volume, inoculum size, pH & temperature)
were kept constant. After incubation period, the quality and texture of the yogurts at different
concentrations of powdered milk were observed and recorded in a table.
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
3.6.5 Effect of Powdered Milk concentration in Water
To study the effect of powdered milk concentration in yogurt production, the fermentation process was
carried out using distilled water. At first, the 500 ml distilled water was measured by a measuring
cylinder and then boiled. After cooling, 100 ml water was taken into five separate plastic containers. To
find out the optimum concentration of powdered milk, five different concentrations of Dano powdered
milk (2%, 4%, 6%, 8% and 10%) were prepared and added to the containers having distilled water and
dissolved properly. 1 tsp. of inoculum was added to each container and they were incubated at 37˚C for
5 hours and 30 minutes. While there were variations among powdered milk concentrations, all other
conditions (milk volume, inoculum size, pH & temperature) were kept constant. After incubation period,
the quality and texture of the yogurts at different concentrations of powdered milk in distilled water
were observed & recorded in a table.
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
CHAPTER: 04
RESULTS
4.1 SPONTANEOUS FERMENTATION
Observation & result:-
After 20 hours of incubation period, the yogurts were observed and their physical characteristics were
recorded in the following table:
Figure 1: Showing yogurts made by spontaneous fermentation
Table 1.1: Characteristics of yogurts made by spontaneous fermentation
Sample
name
Color Aroma Texture Consistency Presence of
water
Red
container
Off-white Slightly
acidic
Smooth Firm, thick +
Yellow
container
White Pleasant
smell
Smooth Firm, thick Absent
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
Microscopic Characteristics:-
(a)
(b)
Figure 2 (a & b): The cell morphology observed under a compound microscope
Table 1.2: Tabular presentation of microscopic characteristics of the cells
Isolates Shape Arrangement Grams
reaction
Presumptive
organism
Isolate no.1
(figure a)
Diplococci Pairs Positive Streptococcus
thermopilus
Isolate no.2
(figure b)
Diplococci Long chain Positive Lactococcus lactis
Isolate no.3
(figure b)
Rod shaped Single Positive Lactobacillus
bulgaricus
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
4.2 INOCULATED FERMENTATION
4.2.1 Observation & result:-
After 22 hours of incubation period, the yogurts were observed and their physical appearances were
recorded in the following table:
Figure 3: showing yogurts inoculated from Shakti & Ultra Doi
Table 2.1: Characteristics of yogurts made by inoculated fermentation
Inoculated
from
Color Aroma Texture Consistency Presence of
water
Shakti doi Off-white Pleasant
smell
Smooth Firm ++
Ultra doi Off-white Pleasant
smell
Smooth Loose, broken +++
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
4.2.1.1 Observation & result:-
After 18 hours of incubation, the yogurts were observed and their physical appearances were recorded in
the following table:
Figure 4: Sub-cultured from spontaneously fermented yogurt (fig.1) & Ultra doi (fig.2)
Table 2.2: Characteristics of yogurts made by inoculated fermentation
Inoculated from Color Aroma Texture Consistency Presence of
water
Spontaneously
fermented
Off-white Slightly
acidic
Rough Thin ++++
Ultra doi
container
White Pleasant
smell
Smooth Firm, thick +
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
Microscopic Characteristics:-
(a) organism from spontaneously fermented yogurt
(b) organism from ultra doi container
Figure 5 (a & b): The cell morphology observed under a compound microscope
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
Table 2.3: Tabular presentation of microscopic characteristics of the cells
Sample name Shape Arrangement Grams reaction Presumptive
organism
Spontaneously
fermented
Long and
short rods
Single Positive Lactobacillus
delbrueckii subsp.
bulgaricus
Ultra doi
container
Rods Single, pairs Positive Lactobacillus
delbrueckii subsp.
bulgaricus
Ultra doi
container
Spiral Single Positive Lactobacillus
delbrueckii subsp.
bulgaricus
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
4.2.1.2 Observation & result:-
After 6 hours of incubation, the yogurts were observed and their physical appearances were recorded in
the following table:
Figure 6: showing continuous fermentation process
Table 2.4: Characteristics of yogurts made by inoculated fermentation
Inoculated
from
Color Aroma Texture Consistency Presence of
water
Green
container
Off-white Pleasant
smell
Smooth Semi-solid ++
Yellow
container
White Slightly
acidic
Smooth Firm, thick Absent
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
Microscopic Characteristics:-
Figure 7: The cell morphology observed under a compound microscope
Table 2.5: Tabular presentation of microscopic characteristics of the cells
Sample name Shape Arrangement Grams reaction Presumptive organism
Green container Rods Short chains Positive Lactobacillus
delbrueckii subsp.
bulgaricus
Yellow container Rods Long chains Positive Lactobacillus
delbrueckii subsp.
bulgaricus
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
4.2.1.3 Observation & result:-
After 5 and 6 hours of separate incubation periods, the yogurts were observed and their physical
appearances were recorded in the following table:
Figure 8: showing yogurts after 5 and 6 hours of incubation period at 37˚C
Table 2.6: Characteristics of yogurts made by inoculated fermentation
Inoculated
from
Color Aroma Texture Consistency Final
pH
Presence of
water
Set A
(5 hrs)
White Slightly
acidic
Smooth Firm, thick 6.3 +
Set B
(6 hrs)
Off-
white
Acidic
smell
Grainy Firm 6.2 ++
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
Microscopic Characteristics:-
Figure 9: The cell morphology observed under a compound microscope
Table 2.7: Tabular presentation of microscopic characteristics of the cells
Sample name Shape Arrangement Grams reaction Presumptive
organism
Set A (5 hrs) Rods Pairs, short chains Positive Lactobacillus
delbrueckii subsp.
bulgaricus
Set B (6 hrs) Rods Dispersed Positive Lactobacillus
delbrueckii subsp.
bulgaricus
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
4.2.B Inoculated fermentation using MRS Agar plate & MRS Broth Culture
Observation & result:-
After 24 hours of incubation, the yogurts were observed and their physical appearances were recorded in
the following table:
Figure 10: Inoculated fermentation of milk using bacterial culture from MRS agar plate
Table 3: Characteristics of yogurts made by using bacterial stock culture
Inoculated
from
Color Aroma Texture Consistency Presence of
water
MRS agar Yellow Unpleasant
smell
Grainy,
pitted
Thin +++
MRS broth Yellow Unpleasant
smell
Irregular Very thin +++
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
4.3 Effect of temperature
Observation & result:-
After 5 hours of incubation period, the effect of different temperatures on milk fermentation was
observed and recorded in the following table:
Figure 11: Effect of temperature at 45˚C (a), 55˚C (b) & 60˚C (c & d) respectively
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
Table 4: Effect of temperature on lactic acid fermentation of milk
Sample
name
Characteristics of yogurts on different temperatures
Color Taste Aroma Texture Consistency Presence of
Water
Set A (45˚C) White Sour Pleasant smell Smooth Moderately
firm
++
Set B (55˚C) Off-white ----- ----- ----- Semisolid with
water
+++
Set C (60˚C) White Sour Pleasant smell Smooth Firm, thick Absent
4.4 Effect of pH
Observation & result:-
After 5 hours of incubation period, the quality and characteristics of the yogurts in different pH were
observed and recorded in the following table:
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
(a) (b) (c)
Figure 12: Effect of pH 5.5 (a), pH 6.5 (b) and pH 8.5 (c) respectively
Table 5: Effect of pH on lactic acid fermentation of milk
Sample
names
Color Aroma Texture Consistency Presence of water Final pH
pH 4.5 ---- ----- ---- ---- ---- ----
pH 5.5 White Sweet
smell
Coarse,
clumps of
yogurt
Thin Water floating
around the clumps of
yogurt
4.7
pH 6.5 Off-white Sweet
smell
Smooth Thick, firm Absent 5.8
pH 8.5 White Smooth Thick, firm Small amount of
water
6.3
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
4.5 Effect of NaCl concentration
Observation & result:-
After 5 hours of incubation period, the quality and characteristics of yogurts in different concentrations
of NaCl were observed and recorded in the following table:
(a)
(b)
Figure 13 (a & b): Effect of NaCl concentrations (0.1 %, 0.5 % and 1.0 %)
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
Table 6.1: Effect of NaCl concentration on lactic acid fermentation of milk
Effect of NaCl concentration
Concentration
of NaCl
Color Aroma Texture Consistency Presence of
water
0.1 % Off-white Sweet smell Smooth,
glossy
Firm, thick Absent
0.5 % Off-white Sweet smell Smooth,
glossy
Firm, thick Absent
1.0 % Off-white Sweet smell Smooth,
glossy
Semi-solid,
loose
Water around
the yogurt
4.5.1 Observation & result:-
After 5 hours of incubation period, the quality and characteristics of 5-set of yogurts in same
concentration of NaCl were observed as well as the changes in pH were recorded in the following tables:
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
(a) (b)
(c)
Figure 14 (a, b & c): yogurts inoculated from 0.1 % NaCl conc. yogurt previously prepared
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
Table 6.2: Effect of NaCl concentration on lactic acid fermentation of milk
Effect of NaCl concentration
Serial
no.
Concentration
of NaCl
Color Aroma Texture Consistency Presence of
water
1 0.1 % White Sweet smell Smooth Semi-solid ++
2 0.1 % White Sweet smell Smooth Firm, thick +
3 0.1 % White Sweet smell Smooth Firm, thick Absent
4 0.1 % White Sweet smell Smooth Firm +
5 0.1 % White Sweet smell Smooth Firm +
Table 6.3: pH of milk measured at 1-hour intervals
Initial pH of milk pH after 1 hour 2 hour 3 hour 4 hour 5 hour
6.5 6.3 6.2 5.4 5.0 4.7
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
4.5.2 Microscopic Observation:-
The cell morphology of Lactobacillus sp. was observed under 100X magnification of a compound
microscope and rod shaped single cells were found.
Figure 15: The cell morphology observed under a compound microscope
Table 6.4: Tabular presentation of microscopic characteristics of the cells
Isolates Shape Arrangement Grams reaction Presumptive organism
Isolate no.1 Coccus Pairs Positive Streptococcus
thermophilus
Isolate no.2 Diplococci Pairs Positive Lactococcus lactis
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
4.6 Effect of powdered milk concentration in liquid milk
Observation & result:-
After 5 hours & 30 minutes of incubation period, the quality and characteristics of yogurts in different
concentrations of powdered milk were observed and recorded in the following table:
Figure 16: Effect of powdered milk conc. (2%, 4%, 6%, 8% and 10%) in liquid milk
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
Table 7: Effect of powdered milk concentration in liquid milk
Concentrations
of PM
Color Aroma Texture Consistency Presence of
water
2 % Off-white Pleasant
smell
Smooth,
glossy
Firm, thick Absent
4 % Off-white Pleasant
smell
Smooth,
glossy
Moderately firm
& thick
+++
6 % Off-white Pleasant
smell
Smooth,
glossy
Moderately firm
& thick
++
8 % Off-white Pleasant
smell
Smooth,
glossy
Firm & thick gel
like
+
10 % Off-white Pleasant
smell
Smooth,
glossy
Very firm, thick
gel like
Absent
*PM= Powdered Milk
4.7 Effect of powdered milk concentration in water
Observation & result:-
After the incubation period, the quality and characteristics of yogurts in different concentrations of
powdered milk were observed and recorded in the following table:
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
(a)
(b)
Figure 17 (a & b): Effect of powdered milk conc. (2%, 4%, 6%, 8% and 10%) in water
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
Table 8: Effect of powdered milk concentration in water
Concentrations
of PM
Color Aroma Texture Consistency Presence of
water
2 % ------- ------- ------- ------ ------
4 % Off-white Pleasant smell ------- Semi-solid,
loose, broken
++++
6 % Off-white Pleasant smell Smooth Semi-solid,
loose
+++
8 % Off-white Pleasant smell Uneven Moderately firm ++
10 % Off-white Pleasant smell Smooth,
glossy
Firm, thick gel
like
+
*PM= Powdered Milk
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
CHAPTER: 05
DISCUSSION
5.1 SPONTANEOUS FERMENTATION
The yogurts made by spontaneous fermentation contained no inoculum in them still they presented good
qualities. They were white in color, had pleasant aroma, smooth texture and firm consistencies. Also, the
presence of water was very limited (figure 1, table 1.1). Moreover, the microscopic observation of the glass
slides showed that the yogurts seemingly contained Streptococcus and Lactobacillus species in them
(figure 2, table 1.2). Therefore, the natural flora of milk triggered the fermentation of lactic acid and as a
result the milk was spontaneously fermented and hence it is called spontaneous fermentation process.
5.2 INOCULATED FERMENTATION
The yogurts inoculated directly from Shakti doi and Ultra doi showed the same characteristics as their
starter cultures, which had a sweet smell and they were semi-solid with water (figure 3, table 2.1). Since,
the inoculum size was large, it did not mix well with the milk and so, it presented no different qualities than
the starter culture (Shakti doi & Ultra doi).
In the consecutive batches of yogurt, the quality and texture indicated massive improvement (table 2.2, 2.4,
2.6). Moreover, the incubation time was shortened in each fermentation batch and therefore, the quality of
yogurt had been improved. In addition, the microscopic characteristics of bacterial cells from every batch
have been observed to ensure that they presumably contained pure culture of Lactobacillus species (figure
5, 7, 9). Further biochemical tests can be performed to confirm their presence.
The stock culture used to inoculate the milk sample was too old and kept at -4˚C for a very long time. At
first, they were transferred to Lactobacillus MRS Agar and MRS broth in order to revive Lactobacillus
species. They took almost 48 hours to grow onto the agar plates and in broth cultures and after that they
were transferred to fresh pasteurized milk and incubated overnight. The yogurts inoculated from the stock
culture presented very poor qualities. The smell was very unpleasant and undesirable, the color was too
yellow, the texture was very grainy and the consistency was semi-solid with water (figure 10, table 3).
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
Therefore, it can be concluded that the corresponding stock culture was contaminated and thus it produced
a very poor quality yogurt which was discarded afterwards.
5.3 Effect of temperature
To find out the optimum temperature for yogurt production, fermentation process was carried out using
three different temperatures (45˚C, 55˚C, 60˚C) and the most suitable and effective temperature was
determined as the optimum temperature for this study.
The yogurt incubated at 45˚C was moderately firm, water was present on top the yogurt and smell was
pleasant (figure 11-a). The second one was incubated at 55˚C and it was semi-solid with water (figure 11-
b). Furthermore, the taste & smell wasn’t pleasant. Thus, it was a very poor quality yogurt. On the other
hand, 60˚C yogurt was the finest and most firm between the other two yogurts. It had a pleasant smell,
smooth texture and thick consistency with no presence of water in the yogurt [figure 11 (c & d), table 4].
Therefore, the optimum temperatures for yogurt production were determined 45˚C and 60˚C respectively.
5.4 Effect of pH
To find out the optimum pH for yogurt production, fermentation process was carried out using four
different pHs (4.5, 5.5, 6.5 & 8.5) and the most suitable and effective pH was determined as the optimum
pH for this study.
At pH 6.5, the most satisfactory quality of yogurt among the four was seen, since it had a sweet smell,
smoother texture, firm consistency and had no presence of water in it (figure 12, table 5). On the contrary,
there were chunks of solid floating in the water in case of pH 5.5 and it could not be classified into a good
quality yogurt in any way. Furthermore, yogurt at pH 8.5 had a smooth texture and thick consistency but
then it had an unpleasant smell. Additionally, when the pH of the milk was decreasing to 4.5, the milk
started to curdle and the pH dropped rapidly to 3.8. Therefore, this part the experiment could not be
performed since the particular milk container gave no results. Consequently, the suitable pH for yogurt
production was determined at pH 6.5 undeniably.
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
5.5 Effect of NaCl concentration
The higher the concentration of NaCl, the less firm the yogurt was. Therefore, yogurt containing 1.0%
NaCl was loose in consistency and gave a watery texture, since salt caused water to come out from the
yogurt. On the other hand, 0.1 % and 0.5 % NaCl concentration in yogurt gave more firm and thicker
consistency as well as smoother texture (figure 13, table 6.1). So, lesser concentration of salt is more
suitable for a good quality yogurt production. Therefore, the optimum concentration of NaCl for yogurt
fermentation was determined at 0.1% and 0.5% NaCl correspondingly.
In subsequent batches of continuous fermentation, the yogurts presented similar physical characteristics
(figure 14, table 6.2) and the pH of milk every 1-hour interval were measured (table 6.3). The microscopic
characteristics of cells isolated from yogurts were observed under compound microscope where they
appeared as cocci shaped arranged in pairs. Presumably, these organisms were Streptococcus species
(figure 15, table 6.4).
5.6 Effect of powdered milk concentration in liquid milk
The higher the concentration of powdered milk was, the thicker and finer the yogurt was. The yogurt
containing 2%, 8% and 10% powdered milk concentration showed thick, firm, smooth gel like consistency
and pleasant aroma (figure 16, table 7). On the other contrary, yogurt having 4% and 6% powdered milk
were moderately firm with the presence of water (table 7). Therefore, the most favorable concentrations of
powdered milk in liquid milk for yogurt fermentation were determined 2%, 8% and 10% respectively.
5.7 Effect of powdered milk concentration in water
The higher the concentration of powdered milk was the thicker and better-quality the yogurt was. The 2%
powdered milk concentration did not show any characteristic of yogurt production and thus fermentation
did not take place (figure 17). Correspondingly, yogurts containing 4% and 6% powdered milk were
semisolid with water. In addition, 8% yogurt was moderately firm and 10% yogurt was thick, firm and
showed a gel like consistency (figure 17, table 8). Therefore, the ideal concentration of powdered milk in
water for yogurt fermentation was determined 10% respectively.
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
CHAPTER: 06
CONCLUSION
In this study, the physicochemical properties affecting lactic acid fermentation of milk were studied and
an optimum condition for a good quality yogurt production has been achieved. Having stated before, the
significant factors that affect the fermentation process are: temperature, pH, NaCl concentration, liquid
and powdered milk concentrations. These factors required for the fermentation process were optimized
and the performance of the fermentation process was enhanced remarkably. The suitable temperature for
lactic acid fermentation was determined 45˚C and 60˚C correspondingly and optimum pH was 6.5;
optimal concentrations of NaCl were achieved 0.1% & 0.5% while, powdered milk concentrations in
liquid milk were 2%, 8% and 10% respectively. In addition, ideal concentration of powdered milk
concentration in water was carefully chosen 10%. The yogurt produced by means of these optimum
conditions showed thick gel-like consistency, smooth texture and contained a pleasant smell which
ensures an efficacious fermentation and they were incubated for up to 5 hours. The cell morphology of
the organisms taken from these yogurts were observed under the compound microscope after performing
Gram’s stain where they appeared as long rod shaped bacteria in pairs and in long chains. Additionally,
some cocco-bacilli were also seen under the microscope which was in pairs and in chains. All these
morphological characteristics confirmed the presence of lactic acid bacteria (LAB) in the corresponding
yogurts. Further biochemical tests are required to confirm the presumptive organisms.
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
CHAPTER: 07
REFERENCES
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Champagne CP and Gardener NJ (2005). Challenges in the Addition of Probiotic Cultures to Foods.
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Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
CHAPTER: 08
APPENDIX
Appendix - A
8.1 MICROBIOLOGICAL MEDIA
Media used in this study, were prepared using methods with appropriate compositions. All media were
sterilized by autoclaving at 121˚C and 15 lb. pressure for 20 minutes.
8.1.1 Composition of Lactobacillus MRS Agar media
Name of the ingredients Amount (Gms/litre)
Proteose peptone 10.00
Beef extract 10.00
Yeast extract 5.00
dextrose 20.00
polysorbate 80.00
Ammonium citrate 2.00
Sodium acetate 5.00
Magnesium sulphate 0.10
Manganese sulphate 0.05
Dipotassium phosphate 2.00
Agar 12.00
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
8.1.2 Composition of Lactobacillus MRS Broth
Name if the ingredients Amount (Gms/litre)
Dextrose 20.00
Proteose peptone 10.00
Beef extract 10.00
Yeast extract 5.00
Sodium acetate 5.00
Ammonium citrate 2.00
Dipotassium phosphate 2.00
Magnesium sulphate 0.10
Manganese sulphate 0.05
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
8.2 REAGENTS & CHEMICALS
There are various reagents that were used for different purposes during the project work. Some of them
were used directly and some were prepared in the lab. The most commonly used reagents and their
composition are shown below:
Serial No. Name of the reagent Manufacturer
1 Crystal violet Sigma, Nentech
2 Distilled water Invitrogen
3 Ethanol (absolute) Merck, Germany
4 Ethanol (70 %)
5 Ethanol (95 %)
6 Gram’s Iodine Solution
7 Hydrochloric Acid (32%) Merck
8 Sodium Hydroxide (1M)
9 Immersion Oil Sigma
10 Lactobacillus MRS Agar Hi Media Laboratories Ltd, India
11 Lactobacillus MRS Broth Hi Media Laboratories Ltd, India
12 Safranin-O Sigma, Loba
13 Sodium Chloride Merck
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
Composition of 1M NaOH
NaOH pellets - 40 g
Distilled water
Composition of Crystal Violet
Crystal violet - 2 g
Ethanol (95%) - 20 ml
Ammonium oxalate - 0.8 g
Distilled water - 80 ml
Composition of Gram’s Iodine Solution
Iodine crystals -1 g
Potassium Iodide -2g
Distilled water -300 ml
Composition of Decolorizer
Acetone - 1 volume
Ethanol (95%) - 1 volume
Composition of Safranin-O
Safranin O - 0.25 g
Ethanol (95%) -10 ml
Distilled water - 90 ml
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
Appendix - B
Instrument name Model no. Origin
Autoclave WAC-47 Daihan Scientific, Korea
Balance machine Core series Adam, UK
Compound microscope Model- CX-21 Olympus
Incubator (37˚C) SAARC Engineering
Incubator (45˚C) Model - DSI300D Taiwan
Laminer airflow cabinet SAARC Engineering
pH meter pHep, HI-98107 HANNA Instruments
Refrigerator Model-0636 Samsung
Vortex mixture VWR International
Water bath Korea
Lactic Acid Fermentation of Pasteurized And Powdered Milk And Optimizing The Factors Affecting
The Fermentation Process
Appendix – C
Serial no. Name of the Glassware and Other
Equipment
Origin
1 Beaker (250 ml) Iwaki, Japan
2 Conical flask (250 ml) Pyrex
3 Measuring cylinder (100 ml & 250 ml) MC, China & Pyrex, UK
4 Glass rod China
5 Glass slide China
6 Petri dishes Germany
7 Screw cap testube (10 ml) Pyrex
8 Reagent bottle (250 ml) with dropper China
9 Inoculating loop India
10 Spatula India
11 Spirit lamp China
12 Bunsen burner Bangladesh
13 Aluminum Foil USA
14 Thermometer China